Method for deprotecting oligonucleotides

Abstract

A method for purifying an oligonucleotide that comprises providing an oligonucleotide attached to a substrate, wherein the oligonucleotide contains phosphate protecting groups; contacting the oligonucleotide with a reagent, e.g., an organic amine, that cleaves the phosphate protecting groups from the oligonucleotide without detaching the oligonucleotide from the substrate; isolating the oligonucleotide attached to the substrate from the cleaved phosphate protecting groups; and cleaving the oligonucleotide from the substrate. This method provides crude oligonucleotide mixtures that are easier to purify and from which the desired full-length oligonucleotide product can be isolated in higher yields.

1. A method for purifying an oligonucleotide that comprises:
 a) providing an oligonucleotide attached to a substrate, wherein the oligonucleotide contains phosphate protecting groups;  
 b) contacting the oligonucleotide with a reagent that cleaves the phosphate protecting groups from the oligonucleotide without detaching the oligonucleotide from the substrate;  
 c) isolating the oligonucleotide attached to the substrate from the cleaved phosphate protecting groups; and  
 d) cleaving the oligonucleotide from the substrate.  
 
     
     
       2. The method of  claim 1 , wherein the substrate is a solid. 
     
     
       3. The method of  claim 1 , wherein the substrate is a liquid. 
     
     
       4. The method of  claim 1 , wherein the substrate is an inorganic material, an organic material, or a combination thereof. 
     
     
       5. The method of  claim 1 , wherein the phosphate protecting group is a group capable of undergoing β-elimination. 
     
     
       6. The method of  claim 5 , wherein the phosphate protecting group is a 2-cyanoethyl group. 
     
     
       7. The method of  claim 1 , wherein the reagent cleaves the phosphate protecting group from the oligonucleotide by β-elimination. 
     
     
       8. The method of  claim 1 , wherein the reagent used to selectively remove phosphate protecting groups is an amine with a formula R—N—R 1 R 2  wherein R, R 1 , and R 2  are independently hydrogen, hydroxy, or a hydrocarbon selected from the group consisting of alkyl, allyl, aryl, cycloalkyl, alkenyl, alkoxy, allyloxy, and aryloxy, and having from one to twenty carbon atoms. 
     
     
       9. The method of  claim 1 , wherein the reagent is an organic amine. 
     
     
       10. The method of  claim 1 , wherein the reagent is diethylamine. 
     
     
       11. The method of  claim 1 , wherein the reagent contains about 20% v/v diethylamine. 
     
     
       12. The method of  claim 1 , wherein the reagent is delivered as a gas. 
     
     
       13. A method of  claim 1 , wherein the oligonucleotide backbone contains at least one phosphodiester linkage. 
     
     
       14. A method of  claim 1  wherein the oligonucleotide backbone contains at least one phosphoramidate linkage. 
     
     
       15. A method for purifying an oligonucleotide that comprises:
 a) providing an oligonucleotide containing a phosphate protecting group attached to a substrate, wherein the phosphate protecting group is 2-cyanoethyl;  
 b) contacting the oligonucleotide with diethylamine to cleave the phosphate protecting groups from the oligonucleotide without detaching the oligonucleotide from the substrate;  
 c)isolating the oligonucleotide attached to the substrate from the cleaved phosphate protecting groups; and  
 d) contacting the oligonucleotide attached to the substrate with ammonium hydroxide to cleave the oligonucleotide from the substrate.